Aroma Investigation of Chios Mastic Gum (Pistacia lentiscus var. Chia

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Chemistry and Biology of Aroma and Taste

Aroma Investigation of Chios Mastic Gum (Pistacia lentiscus var. Chia) using Headspace Gas Chromatography Combined with Olfactory Detection and Chiral Analysis Marina Rigling, Marco Alexander Fraatz, Stefan Trögel, Jinyuan Sun, Holger Zorn, and Yanyan Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00143 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Journal of Agricultural and Food Chemistry

Aroma Investigation of Chios Mastic Gum (Pistacia lentiscus var. Chia) using Headspace Gas Chromatography Combined with Olfactory Detection and Chiral Analysis

Marina Rigling†, Marco Alexander Fraatz‡, Stefan Trögel§, Jinyuan Sun¥, Holger Zorn‡, Yanyan Zhang*† †University of Hohenheim, Institute of Food Science and Biotechnology, Department of Flavor Chemistry, Fruwirthstraße 12,

70599 Stuttgart, Germany ‡Justus

Liebig University Giessen, Institute of Food Chemistry and Food Biotechnology, Heinrich-Buff-Ring 17, 35392 Giessen,

Germany §Justus

Liebig University Giessen, Institute of Veterinary Food Science, Frankfurter Straße 92, 35392 Giessen, Germany

¥Beijing

Technology and Business University, Beijing Key Laboratory of Flavor Chemistry, Fucheng Road 11, 100048 Beijing,

China

*Corresponding author Yanyan Zhang, Tel: +49 711 459-24871, Fax: + 49 711 459-24873, E-mail: [email protected]

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ABSTRACT

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Chios mastic gum (Pistacia lentiscus var. Chia) exhibits an intensely sourish, green, resinous,

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and woody odor note with hints of citrus and pine. Despite of its attractive flavor, no description

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of its aroma properties by molecular sensory techniques has been published so far. Twenty-five

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odor-active compounds with flavor dilution (FD) factors of 1 to 512 were identified by gas

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chromatography-mass spectrometry-olfactometry (GC-MS-O) combined with headspace solid

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phase microextraction (HS-SPME) and stir bar sorptive extraction (HS-SBSE). Quantitative

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analyses performed by multiple HS-SPME and calculation of odor activity values of ten odorants

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with high FD factors revealed an essential role of several minor components (e.g., -myrcene,

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limonene, -linalool, and perillene) for the overall aroma of mastic gum besides the dominating

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compound -pinene. The indispensable contribution of the minor odorants to mastic gum was

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further confirmed by aroma recombination and omission tests. Varying enantiomeric excess

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values of the key odorants were observed by multidimensional GC-MS.

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Keywords: aroma, Chios mastic gum, HS-SPME, HS-SBSE, MHS-SPME, GC-O, MDGC-MS


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INTRODUCTION

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Pistacia lentiscus L (mastic tree) belongs to the Anacardiaceae family and is an evergreen shrub

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distributed in the Mediterranean area. The var. Chia is grown almost exclusively in the southern

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part of Chios Island, Greece. Chios mastic gum derived from mastic tree is a semitransparent,

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white to yellowish natural resin.1,2 It is traditionally used for the treatment of digestive, hepatic,

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and kidney diseases since at least 3,000 years. The pharmaceutical properties of mastic gum (e.g.,

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antimicrobial, anti-inflammatory, and antiseptic activities) are mainly related to triterpenoid

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compounds.3,4 Mastic gum and its essential oil are extensively used in numerous foods (e.g.,

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biscuits, ice cream, chewing gum, mastic “sweets of the spoon”, and soft drinks), perfume, and

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cosmetics.5-7 Apart from its functional properties, mastic gum is characterized by a pleasant,

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intensely sourish, green, resinous, and woody odor with hints of citrus and pine. As a unique and

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special spice with approximately 250 tons annual export from Chios Island, Chios mastic gum is

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of enormous locally economic importance.8 So far, various terpenes have been identified in the

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volatile fraction of Chios mastic gum. Among them, -pinene (41%-67% of total peak area)

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represents the major constitute of Chios mastic essential oil produced by hydrodistillation or

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supercritical CO2 extraction.9-12 Although the chemical composition of Chios mastic gum has

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been comprehensively analyzed, the molecular basis of its favorable aroma properties remains to

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be elucidated.

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Being fast, solvent-free, sensitive, and reliable techniques, headspace solid phase microextraction

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(HS-SPME) and headspace stir bar sorptive extraction (HS-SBSE) represent powerful tools for

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aroma analysis.13-17 Nevertheless, a quantitative analysis of volatiles of solid samples by HS-

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SPME and HS-SBSE is challenging because of matrix interferences caused by non-exhaustive

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extraction.17-21 Therefore, multiple HS-SPME (MHS-SPME) has been developed as an attractive 3 ACS Paragon Plus Environment


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and reliable approach for quantitation of volatiles especially from solid samples. HS-SPME is

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performed repeatedly, which allows estimating the correlation between the total peak area and the

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original amount of an individual analyte present in the sample. The specific equilibrium of an

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analyte in the three-phase system (fiber coating/headspace/sample matrix) is established in every

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extraction step.22 The total peak area of an exhaustive extraction of each analyte can be calculated

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from the peak areas obtained from the individual extractions. Hence, the quantitative analysis by

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MHS-SPME becomes independent from the sample’s matrix.18-21

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A comprehensive analysis of the aroma profile of Chios mastic gum may disclose the

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contribution of key odorants to its overall favorable flavor attributes, and will also allow for an

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efficient quality control of mastic gum used as flavoring in food and cosmetics. The aroma

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profile of mastic gum was, therefore, comprehensively decoded by means of molecular sensory

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science in the present study. This implied (i) the identification of key odorants by aroma dilution

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analyses (ADAs) after HS-SPME and HS-SBSE, (ii) chiral analysis of the key odorants by means

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of multidimensional gas chromatography-mass spectrometry (MDGC-MS), (iii) quantitation of

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the key odorants with high flavor dilution (FD) factors by MHS-SPME, (iv) calculation of odor

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activity values on the basis of the respective odor thresholds, and (v) validation of the analytical

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results by aroma recombination and omission studies.

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MATERIALS AND METHODS

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Materials and Chemicals.

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Mastic gum was harvested in July 2016 in Chios and as a commercial product provided by the

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Chios Gum Mastic Growers Association (Chios, Greece). It was stored at -20 °C until aroma

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analysis.


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Acetic acid (98%), acetone (100%), -pinene (99%), and ethanol (99.8%) were obtained from

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Carl Roth (Karlsruhe, Germany). Z/E-Carveol (95%), limonene (97%), myrtenal (98%),

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-terpinene (97%), -pinene (98%), benzyl methyl ether (98%), and an alkane series of C7-C30

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(100 µL each) were obtained from Sigma-Aldrich (Steinheim, Germany). (R)-Camphene (90%)

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and (R)--pinene (97%) were purchased from Merck (Darmstadt, Germany). (S)-Limonene

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(97%), oct-1-en-3-one (97%), and (S)--pinene (99%) were obtained from Alfa Aesar (Karlsruhe,

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Germany). -Linalool (97%), (R)-linalool (95%), 2-nonanone (99%), -myrcene (90%), terpinen-

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4-ol (97%), and (S)-Z-verbenol (97%) were purchased from Acros Organics (Geel, Belgium).

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(R)-Terpinen-4-ol (98%) and 2-methoxytoluene (99%) were bought from Fisher Scientific

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(Schwerte, Germany). Camphene (80%) was bought from TCI (Eschborn, Germany). Perillene

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(98%) was obtained from Chengdu Push Bio-Technology Co Ltd. (Chengdu, China). Hexane

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(HPLC-grade) was purchased from Th. Geyer (Renningen, Germany). Methanol (HPLC grade)

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was obtained from VWR (Darmstadt, Germany). For gas chromatography, helium 5.0 (Praxair,

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Duesseldorf, Germany) and nitrogen 5.0 (Linde, Munich, Germany) were used.

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Headspace Solid Phase Microextraction (HS-SPME).

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For HS-SPME a CAR/PDMS/DVB fiber (carboxene/polydimethylsiloxane/divinylbenzene,

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30/50 µm, 1 cm fiber length) and a CAR/PDMS fiber (carboxene/polydimethylsiloxane, 85 µm, 1

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cm fiber length) (Supelco, Steinheim, Germany) were tested. Prior to aroma analysis, Chios

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mastic gum was frozen in liquid nitrogen and powdered by a pestle. Forty mg of mastic gum

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powder was transferred into a headspace (HS) vial (20 mL). The samples were agitated for 10,

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20, and 30 min (500 rpm) at 35, 45, and 55 °C, followed by headspace extraction at the same

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temperature as during incubation for 25, 35, and 45 min. Afterwards, the analytes were directly

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desorbed in the split/splitless inlet at 250 °C using an SPME liner of a gas chromatography 5 ACS Paragon Plus Environment


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system equipped with a tandem mass spectrometry detector and an olfactory detection port (GC-

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MS/MS-O) for 1 min. After desorption, the fiber was cleaned under the conditions recommended

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by the manufacturer.

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Headspace Stir Bar Sorptive Extraction (HS-SBSE).

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For HS-SBSE analysis, 40 mg of powdered mastic gum was added into a HS-vial. A ten-

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millimeter stir bar with 0.5 mm PDMS coating (Gerstel, Muelheim an der Ruhr, Germany) was

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fixed in the headspace of the vial using a magnet. The volatiles were extracted in a water bath

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adjusted to 45 °C for 75 min. After extraction, the stir bar was removed with forceps, rinsed with

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deionized water to remove whirled dust from the sample, dried, and placed in a thermal

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desorption unit (TDU) liner (Gerstel). Desorption started at 40 °C (0.5 min), and the temperature

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was then ramped at 120 °C/min to 250 °C and held for 10 min. Cryofocusing was performed in a

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cooled injection system (CIS) (Gerstel) equipped with a Tenax liner (Gerstel) with solvent-vent

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mode (40 mL/min). The analytes were released to a gas chromatography system equipped with a

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mass spectrometric detector and an olfactory detection port (GC-MS-O) system. The start

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temperature was -100 °C and was increased with 12 °C/min to 250 °C, and finally held for 8 min.

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Gas Chromatography (GC).

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HS-SPME analysis was carried out on a GC-MS/MS-O system (in single quadrupole mode). HS-

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SBSE analysis was performed on a GC-MS-O system equipped with a thermal desorption unit

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(TDU) and cooled injection system (CIS). The basic parameters for GC-MS/MS-O and GC-MS-

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O systems were the same as described by Zhang et al. (2014)15 and Trapp et al. (2018)17,

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respectively. In detail, a polar Agilent J&W VF-WAXms column (30 m × 0.25 mm i.d. × 0.25

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µm film thickness) and a non-polar J&W DB5ms column (30 m × 0.25 mm i.d. × 0.25 µm film

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thickness) were used for both GC systems (Agilent Technologies, Waldbronn, Germany). Helium 6 ACS Paragon Plus Environment

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(5.0) (Paxair, Duesseldorf, Germany) served as carrier gas with a constant flow rate of 1.65

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mL/min. The gas flow was split 1:1 into the MS detector and the ODP by means of a

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µFlowManager Splitter (Gerstel) with a column outlet pressure of 20 kPa. The GC oven

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temperature was held at 40 °C (3 min), then ramped with 5 °C/min to 240 °C (10 min). The

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following parameters were applied: MS mode, scan; scan range, m/z 40 – 330; electron ionization

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energy, 70 eV; source temperature, 230 °C; quadrupole temperature, 150 °C; MS/MS transfer

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line, 250 °C only for the GC-MS/MS-O system; ODP 3 transfer line temperature, 250 °C; ODP

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mixing chamber temperature, 150 °C; ODP 3 makeup gas, N2 (5.0) (Linde, Munich, Germany).

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The GC-O experiments were conducted by one trained person. The original sample and all

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dilution steps were smelled at least in triplicate.

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Aroma Dilution Analysis (ADA).

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For HS-SPME and HS-SBSE revised ADAs were performed by increasing the GC inlet split

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ratios15,23 and the CIS inlet split ratios17, respectively. Due to the limit of the split ratios of the

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GC-MS/MS-O system (maximum split ratio 256), the amount of sample was halved to achieve

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FD factors of up to 512 in HS-SPME-ADA analysis.

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Multidimensional GC-MS (MDGC-MS).

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Chiral analysis of the key odor-active compounds of Chios mastic gum was performed with a

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MDGC-MS system (Shimadzu, Duisburg, Germany). GC 1, a Shimadzu GC 2010 Plus was

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coupled to a flame ionization detector (FID). GC 2, a Shimadzu GC 2010 Plus was coupled with

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a mass spectrometry detector (GC-MS-QP2010). GC 1 was connected via a Multi Dean’s Switch

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(MDS) to GC 2. Manual HS-SPME injections were carried out.



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GC 1. Helium (5.0) was used as carrier gas at a constant flow rate of 2.06 mL/min. A polar

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Agilent J&W VF-WAXms column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) (Agilent

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Technologies, Waldbronn, Germany) was used for separation of the target compounds. The

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operational conditions were as follows: constant inlet pressure 235.3 kPa; inlet temperature 250

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°C; splitless 2 min; initial linear velocity 25 cm/s; temperature program: 40 °C (3 min), ramped at

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5 °C/min to 220 °C for 6 min. FID parameters: 250 °C; H2 flow 40 mL/min; air flow

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400 mL/min; make-up gas N2 (5.0) 30 mL/min.

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GC 2. For the separation of enantiomers, a BGB-176 column (30% 2,3-dimethyl-6-tert-butyl-

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dimethylsilyl--cyclodextrin in 15% phenyl-, 85%-polymethylsiloxane, 30 m × 0.25 mm i.d. ×

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0.25 µm film thickness) (BGB Analytics, Rheinfelden, Germany) was used. The parameters were

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as follows: transfer line temperature GC 1 and GC 2 200 °C; linear velocity 25 cm/s; switching

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pressure 156.4 kPa; electron ionization energy 70 eV; source temperature 200 °C; quadrupole

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temperature 150 °C; MS transfer line temperature 220 °C. For the analysis of -pinene, -pinene,

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limonene, terpinen-4-ol, and Z-verbenol, the oven temperature program was set as following: 40

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°C (3 min), ramped at 1 °C/min to 150 °C (3 min), and then ramped at 20 °C/min to 200 °C (3

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min). The oven temperature program for the analysis of camphene and -linalool held 40 °C

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(3 min), increased at 5 °C/min to 60 °C (40 min), ramped at 20 °C/min to 95 °C (40 min) and

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then ramped with 20 °C/min to 200 °C (3 min). The cutting windows were set in the range of 15

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to 30 sec according to the peak widths of the odorants. The data were collected using the

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Shimadzu MDGC solution software 1.01.

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Compound Identification.


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The odor-active compounds were identified by their characteristic odor, the retention indices (RI)

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on two columns of different polarity (VF-WAXms and DB-5ms), and the mass spectra in

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comparison with those of authentic standards and data published in literature.7,10-12

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Quantitative Analysis by Means of Multiple HS-SPME (MHS-SPME).

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Ten key odorants were quantified by means of MHS-SPME. The FD factor, a quantifier ion, and

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two qualifier ions of each odorant are shown in Table S1. The compounds were quantified by

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four consecutive extractions of the headspace.18

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The peak area of the ith extraction is described by Equation 1. 𝐴𝑖 = 𝐴1𝛽𝑖 ― 1

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(1)

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With Ai indicating the peak area of the ith extraction, A1 the peak area of the first extraction, and

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 a constant representing the extractability of each compound.  is calculated by the linear

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regression of ln Ai against (i-1)th extraction.

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Via the peak areas obtained from consecutive extractions, the theoretical total peak (AT) is then

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calculated by Equation 2.

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𝐴1

∞

𝐴𝑇 = ∑𝑖 = 1𝐴𝑖 =

(1 ― 𝛽)

(2)

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The calibration factors are obtained from external calibration graphs, constructed with standard

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compounds by MHS-SPME, and the concentrations of the key odorants present in Chios mastic

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gum are calculated from their respective AT.

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Optimization of Amounts of Chios Mastic Gum. 9 ACS Paragon Plus Environment


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Different amounts (10, 20, 30, and 40 mg) of mastic gum powder were added into HS-vials and

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sealed with caps, respectively. Four consecutive extractions under optimum HS-SPME conditions

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were carried out. The proper amount of mastic gum powder was chosen based on the linear

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regression analyses of the peak areas of key odorants obtained in the consecutive measurements.

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Establishment of Calibration Curves of Standard Aqueous Solutions with Key Odorants.

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The calibration curves of aqueous standard solutions with the corresponding key odorants were

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established to calculate the calibration factors. The standard substances were dissolved in

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methanol (Table S2) and then stepwise-diluted with distilled water (1:10, 1:25, 1:50, 1:100,

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1:250, and 1:500). For external standard calibration, 100 µL of each dilution was transferred into

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a HS-vial and then analyzed by MHS-SPME under the same conditions as described above. The

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calibration curve for each target compound was determined by plotting the total peak area against

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the corresponding amount. For each calibration curve, the regression correlation coefficient (R2)

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and ranges (R) were calculated based on duplicate analyses.

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Calculation of Odor Activity Values (OAVs).

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OAVs were calculated from the concentrations determined in Chios mastic gum and the

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respective odor thresholds in water reported in the literature.24-29 The odor thresholds of

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camphene and Z-verbenol were determined as described by Cancho et al.30 due to lack of

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available data in the literature.

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Aroma Recombination.

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Based on quantitative analytical data of ten odorants, an aroma recombinate was prepared with

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water as matrix. Six odor attributes (sourish, green, citrus-like, pine-like, resinous, and woody)

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were defined for description of the overall aroma profile of Chios mastic gum and the 10 ACS Paragon Plus Environment

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corresponding aroma model. A unipolar five-point scale (0 to 5; 0 not detectable; 1 weak; 3

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moderate; 5 strong) was used to express the odor intensities. The values were given by a sensory

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panel consisting of 20 experienced assessors (13 females, 7 males; all non-smokers; mean age

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24). All assessors have participated in a sensory training hold at the Institute for Food Chemistry

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and Food Biotechnology at Justus-Liebig University Giessen for two weeks.

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Omission Tests.

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Five aroma models were prepared by omitting a single compound (-pinene, -myrcene,

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limonene, -linalool or perillene) from the complete recombinate. The given odor qualities of the

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reduced models and the complete recombinate were compared using triangle tests.31,32 The

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sensory panel for the omission tests was the same as the one described above.

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Statistical Analysis.

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Analysis of variance (ANOVA) was conducted to examine the aroma recombination study

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(p > . For the performed triangle tests in the omission tests, the significance of the difference

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 detected was calculated according to Jellinek (1985).32

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RESULTS AND DISCUSSION

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Identification of Potent Odor-Active Compounds of Chios Mastic Gum.

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The aroma of Chios mastic gum is unique and pleasant, and is perceived as sourish, green,

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resinous, and woody odor with hints of citrus and pine. Each extraction procedure offers specific

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advantages, but also has particular drawbacks under certain circumstances on the other side.33

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Therefore no single isolation method can picture the entire aroma profile of a sample. To

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elucidate essential correlations between key odorants and the aroma attributes of Chios mastic 11 ACS Paragon Plus Environment


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gum, the volatiles were isolated by HS-SPME and by HS-SBSE to get a representative headspace

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aroma profile.

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Key Odor-Active Compounds Identified by HS-SPME-GC-MS/MS-O.

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Optimization of HS-SPME.

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Many studies have shown that the SPME parameters, including incubation time, extraction time,

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extraction temperature, and fiber type, greatly affect the number and intensity of aroma

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compounds perceived by GC-O from the respective matrix.14,15 To comprehensively analyze the

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aroma profile of Chios mastic gum, the SPME parameters were systematically optimized based

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on compounds that have been reported as volatiles of mastic gum previously.7,10-12 The extraction

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efficiency was expressed as percentage of the peak area of each compound relative to the

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maximum peak area observed under varying conditions .

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The highest extraction efficiency for most volatiles was observed with an incubation time of

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10 min (Figure S1A). A longer incubation time led to a decrease of the peak areas of -pinene,

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camphene, -pinene, limonene, and 2-nonanone. Only two analytes (-linalool and Z-verbenol)

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showed a slight increase with longer incubation times (30 min). Hence, an incubation time of 10

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min was chosen for further experiments.

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The extraction of camphene, -pinene, limonene, 2-nonanone, and -linalool was optimal at an

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extraction time of 45 min while the peak areas of Z-verbenol significantly decreased at extraction

229

times of > 25 min (Figure S1B). Considering the extraction efficiency for all selected volatiles,

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further experiments were performed with an extraction time of 45 min.

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The maximum peak area of highly volatile compounds (α-pinene, β-pinene, camphene, and

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limonene) was obtained at an extraction temperature of 35 °C (Figure S1C). At increased 12 ACS Paragon Plus Environment

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extraction temperatures (45 °C and 55 °C), the extraction efficiency for 2-nonanone, -linalool,

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and Z-verbenol with comparatively high boiling points was improved. However, liquefaction of

235

mastic gum was observed at 55 °C, which might distort the original aroma profile. Therefore, an

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extraction temperature of 45 °C was chosen as a compromise.

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Bipolar, carbon-based fibers such as CAR/PDMS and CAR/PDMS/DVB have been successfully

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applied to adsorb compounds with various polarities and volatilities in e.g. orange juice14,

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fermented wort15, truffles16, and soy sauce23. With a CAR/PDMS/DVB fiber, 23 odor-active

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compounds were trapped from Chios mastic gum, whereas only 15 compounds were perceived

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after HS-SPME extraction using a CAR/PDMS fiber (Table S3). Thus, the CAR/PDMS/DVB

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fiber was selected for trapping key odorants of Chios mastic gum.

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Identification of Odor-Active Compounds.

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Using the optimized HS-SPME conditions, 23 odor-active regions from Chios mastic gum were

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perceived at the ODP, and 18 corresponding odorants were identified or tentatively identified (*)

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by GC-MS/MS-O analysis (Table 1). They included 4 monoterpenes (mono- and bicyclic:

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-pinene, -pinene, camphene, and limonene), 5 monoterpenoids (monoterpene alcohols:

248

E,E-5-caranol*, -linalool, Z-verbenol, and carveol as well as the monoterpene aldehyde

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myrtenal), one sesquiterpene-epoxide (humulene-1,2-epoxide*), three aliphatic ketones (acetone,

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oct-1-en-3-one, and 2-nonanone), one aliphatic carboxylic acid (acetic acid), one aliphatic

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alcohol (ethanol), one aliphatic hydrocarbon (6Z-2,6-dimethyl-2,6-octadiene*), and two ethers (2-

252

methoxytoluene and isothymol methyl ether*). Five compounds were still unknown: #14 (a

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green, pea- and citrus-like odor), #15 (a woody, and green coffee-like odor), #22 (an earthy

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odor), #26 (sweetish, and marzipan-like odor), and #27 (an earthy odor). All identified

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compounds occurred as single compounds without co-elution, apart from 2-nonanone (#12). 213 ACS Paragon Plus Environment


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Nonanone co-eluted with benzyl methyl ether. Obviously, the odor impression of 2-nonanone

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matched well the aroma (#12, fresh, green, resinous) perceived from Chios mastic gum, whereas

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benzyl methyl ether exhibited an artificial and rubber-like odor.

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Determination of Key Odor-Active Compounds.

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By application of a revised aroma dilution analysis by increasing the GC inlet split ratios15,23, 23

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odor impressions were detected with flavor dilution (FD) factors of 1-512 (Table 1). An unknown

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compound (#14) was determined with the highest FD factor (512) followed by α-pinene (FD

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256), β-linalool (FD 256), 2-nonanone (FD 128), β-pinene (FD 128), camphene (FD 64), an

264

unknown compound (# 22, FD 16), Z-verbenol (FD 8), limonene (FD 8), and humulene-1,2-

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epoxide (FD 8).

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Key Odor-Active Compounds Identified by HS-SBSE-GC-MS-O with TDU.

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Identification of Odor-Active Compounds.

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Since the odorant with the highest FD factor perceived after HS-SPME was still unknown, HS-

269

SBSE was applied as a second extraction method. The larger surface of the twister compared to

270

SPME fibers results in higher sensitivity of HS-SBSE,17,34,35 which may enable HS-SBSE to trap

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key odorants that have not been identified after HS-SPME. On the other side, HS-SBSE is

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applied in combination with a cooled injection system (CIS). The CIS represents a precise,

273

gentle, and discrimination-free injection technique and results in sharp peaks on GC columns.34

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Thirteen odor-active regions were perceived from Chios mastic gum by HS-SBSE-GC-MS-O

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with TDU analysis (Table 1). They included five monoterpenes (-pinene, -pinene, camphene,

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-myrcene, and -terpinene), three monoterpene alcohols (-linalool, E-verbenol, and Z-

277

verbenol*), two monoterpene carbonyls (-campholenal* and -campholenal*), one furan 14 ACS Paragon Plus Environment

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monoterpene (perillene), one aliphatic ketone (oct-1-en-3-one), and one carboxylic acid (acetic

279

acid). All compounds occurred as single compounds without co-elution. Although the number of

280

odor impressions perceived by HS-SBSE-GC-MS-O with a TDU system (13 compounds) was

281

much less than those perceived by HS-SPME-GC-MS/MS-O (23 compounds), four new odor

282

impressions were sniffed (#8, #10, #17, and #19) and four terpenes (-myrcene, -terpinene,

283

-campholenal, and E-verbenol) were identified. These results suggest that the adsorbent

284

(PDMS) as well as the larger phase volume (24 µL) of the stir bar improved the extraction of

285

non-polar terpenes34,35, which were not trapped by SPME with the CAR/PDMS/DVB fiber. More

286

importantly, some unknown odorants perceived after HS-SPME could be identified by HS-SBSE.

287

A better separation of odor impressions #14 and #15 was achieved by HS-SBSE-GC-MS-O using

288

a TDU. The most important odorant perceived after HS-SPME was identified as perillene (#14).

289

Additionally, the unknown compounds (#15 and #22) were characterized as -campholenal and

290

terpinen-4-ol. Unfortunately, the volatiles responsible for the odor-active regions (#26 and #27)

291

could not be identified.

292

Determination of Key Aroma Compounds.

293

By application of a revised aroma dilution analysis by increasing the CIS inlet split ratios17 13

294

odor impressions were detected with FD factors of 1-128 (Table 1). Perillene and -pinene with

295

the highest FD factors (FD 128) were confirmed as the most important odorants of Chios mastic

296

gum. Surprisingly, β-myrcene with an FD factor of 128 also essentially contributed to the aroma

297

of mastic gum, though it was not detected by HS-SPME (Table 1). Besides these three potent

298

odorants, -linalool, -pinene, terpinen-4-ol, camphene, and E-verbenol possessed FD ≥ 8 as

299

well.



300

In total, 25 odorants of Chios mastic gum were identified after headspace microextraction by

301

SPME and SBSE, and only 16 of these compounds have been reported in the literature previously

302

(Table 1).7,10-12 Overall, a comprehensive aroma spectrum of Chios mastic gum was obtained by

303

combining SPME and SBSE extraction. Twelve compounds, namely -pinene, -pinene,

304

camphene, -myrcene, limonene, 2-nonanone, perillene, -linalool, E-verbenol, terpinen-4-ol,

305

Z-verbenol, and humulene-1,2-epoxide were considered as key odorants of Chios mastic gum

306

based on their FD factors ≥ 8.

307

Chiral Analysis of Key Odor-Active Compounds by MDGC-MS.

308

Due to the differing sensory qualities of enantiomers, the optical purities of the key odorants

309

identified in Chios mastic gum were analyzed by means of MDGC-MS. The optical purities of

310

perillene and E-verbenol could not be determined due to lack of commercially available

311

enantiomerically pure standards. The separation of the enantiomers of selected odorants by

312

MDGC-MS is shown in Figure 1.

313

High enantiomeric excess (ee) values were observed for -pinene (99.2% ee for (R)--pinene),

314

-linalool (91.3% ee for (S)--linalool), and Z-verbenol (98.3% ee for (S)-Z-verbenol) (Table 2).

315

The odor description of -pinene observed in mastic gum matched well the odor of (R)--pinene

316

which was reported as pungent, resinous, and minty. High ee values for (R)--pinene found in

317

different Chios mastic products were also reported by Paraschos et al. (2016)36. The odor of (S)-

318

-linalool has been described as floral, green, and sweetish, whereas an intensely woody and

319

lavender-like odor note was reported for the (R)-enantiomer.28 The odor impression of -linalool

320

(a floral, fresh, and fruity odor) perceived from mastic gum is in good agreement with the odor

321

description of (S)--linalool. Although -linalool present in natural mastic gum showed a high ee 16 ACS Paragon Plus Environment

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value of > 90% for the (S)-enantiomer (Table 2), racemic -linalool was detected in Chios mastic

323

water.37 Mastic water is prepared during the steam distillation of mastic gum for the production

324

of mastic essential oil.37 Presumably, the enantiomeric distribution of -linalool was changed

325

during hydrodistillation under thermal stress.38 Z-Verbenol was perceived as intensely pine-like,

326

which supported the odor impression of the (S)-enantiomer reported by Boelens et al. (1993).28

327

The enantiomeric purity of Z-verbenol in Chios mastic gum is reported here for the first time.

328

The ee values of -pinene, camphene, limonene, and terpinen-4-ol ranged from 25% to 56%

329

(Table 2). (R)--Pinene imparts a pine-like and resinous odor note while a herbal and resinous

330

odor was described for the (S)-enantiomer.39 The enantiomeric purity of -pinene detected in

331

mastic gum well reflects the sniffed resinous and fresh odor impression. In our study, the odor

332

impressions of (R)-camphene and the racemic mixture differed remarkably. (R)-Camphene

333

possessed a pungent, pine-like, and camphor-like odor whereas an additional sweetish and fruity

334

note was perceived from the racemate. The (S)-camphene enantiomer might thus exhibit a

335

sweetish and fruity odor note. The dominating pungent odor impression of camphene sniffed

336

from mastic gum was in good agreement with the enantiomeric excess of the (R)-enantiomer.

337

(S)-Limonene has a turpentine- and slightly citrus-like aroma, while the (R)-enantiomer was

338

reported to smell fresh, citrus- and orange-like.28 The results of the olfactory analysis of limonene

339

(sugary, citrus-like, and fruity) might be attributed to the lower odor threshold of the (R)-

340

enantiomer (200 µg/kg) compared to the (S)-enantiomer (500 µg/kg)28, though higher amounts of

341

the (S)-enantiomer were detected in mastic gum. For terpinen-4-ol, similar odor impressions of

342

the (R)- and (S)-enantiomers were described as warm, and earthy40, which is consistent with the

343

results of Chios mastic gum detected at the olfactory detection port.



344

Quantitative Analysis of Key Odor-Active Compounds of Chios Mastic Gum by Multiple HS-

345

SPME (MHS-SPME).

346

All MS signals (extracted ion chromatograms, EIC) of the 12 key odor-active compounds (FD ≥

347

8) apart from E-verbenol were detectable in the GC-MS/MS-O total ion current chromatogram

348

after HS-SPME of Chios mastic gum. As no authentic standards of humulene-1,2-epoxide (FD 8)

349

and E-verbenol (FD 8) were commercially available, these compounds could not be quantitated.

350

The quantitation of the remaining ten key odorants of Chios mastic gum was performed by

351

multiple headspace extraction.

352

The quantitation of volatiles of solid and liquid samples by means of MHS-SPME has been

353

validated by comparison to stable isotopic dilution analyses (SIDA) e.g. for roasted coffee

354

powder and wine.41,42 Comparable performance parameters to SIDA were achieved for accuracy,

355

sensitivity, and repeatability. Apart from that, MHS-SPME has been successfully applied for the

356

quantitation of volatiles of mushrooms19, tomatoes20, sausages43, and bread44. A few studies have

357

reported on relative amounts of volatiles from mastic gum in form of GC-area%,7,10-12 and

358

Daferera et al. (2002) quantified -pinene and -myrcene in Chios mastic oil by means of FT-

359

Raman spectroscopy.2 In the present study, the key volatiles of Chios mastic gum have been

360

comprehensively quantified by means of MHS-SPME for the first time.

361

The independence from matrix effects is a remarkable merit to accurately quantitate volatiles by

362

means of MHS-SPME. To this end, a linear decrease of the logarithm (ln) of the peak area of

363

each volatile compound with the headspace extraction steps has to be observed. Previous studies

364

have shown that the amount of the sample used for quantitation is the key factor to obtain a linear

365

relationship of the ln of the peak area of each volatile compound with the headspace extraction

366

step during the consecutive extractions.18-21 Hence, four consecutive extractions were carried out 18 ACS Paragon Plus Environment

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367

with different amounts of mastic gum powder (10, 20, 30, and 40 mg). Overall, 10 mg of mastic

368

gum showed the best linear decrease for all compounds (R2 > 0.935), with the exception of –

369

pinene and Z-verbenol (Table 3). When the amounts of mastic gum powder were adjusted to ~ 1

370

mg and ~ 35 mg respectively, high correlation coefficients for -pinene and Z-verbenol were

371

obtained (R2 = 0.962 and R2 = 0.952). Using the optimal amount of mastic gum powder, the 

372

value of each key odorant was calculated according to Equation (1). Figure 2 shows the linear

373

plots for the calculation of  values of four selected key odorants. For all analytes, the  values

374

ranged from 0.56 and 0.90 (Table 4), which is within the valid range of  values (0.40 – 0.95).20

375

These results demonstrated that the MHS-SPME approach developed here was feasible and

376

reliable for the quantitation of the key odorants of Chios mastic gum. Linear regression

377

calculation was applied to obtain the y-intercept A1*. To avoid the random variation of the

378

experimental first peak area (A1), the y-intercept (A1*) was used for calculation of the theoretical

379

total peak area (AT) in our study. With determined  value and the intercept A1*, theoretical AT of

380

each key odorant in mastic gum was calculated using Equation (2) (Table 4).

381

Afterwards, a series of aqueous standard solutions were prepared to establish calibration curves

382

of the total peak areas versus the masses of ten key odorants. The obtained correlation coefficient

383

of each compound was acceptable (R2 ≥ 0.965) in the defined linear range (Table S4). The total

384

peak area of each key odorant in mastic gum powder was interpolated into the calibration curves

385

and their concentrations were finally calculated (Table 5). By means of MHS-SPME analyses 10

386

key odorants were quantified with a degree of precision (relative standard deviation, RSD
1 and

393

thus contributed to the characteristic aroma of mastic gum. As a dominating volatile of mastic

394

gum,7,10-12 -pinene (10,000 µg/g, a forest-like and resinous odor) was considered as the most

395

important characteristic odorant of Chios mastic gum (OAV 1,700,000). Surprisingly, several

396

minor components like -myrcene (200 µg/g), limonene (99 µg/g), -linalool (44 µg/g), and

397

perillene (130 µg/g) also exhibited high OAVs (OAVs > 1500), which emphasized their

398

important impact on the overall aroma of Chios mastic gum (Table 5).

399

Aroma Recombination and Omission Tests.

400

OAVs well reflected the contribution of single key odorants to the overall aroma of Chios mastic

401

gum. However, it is not possible to illustrate how interactions of key odorants impact the odor of

402

the sample due to limitations of the OAV concept.45 To finally proof the typical Chios mastic

403

gum aroma, aroma recombination and omission experiments were performed.

404

Six sensory descriptors, namely sourish, green, citrus, pine, resinous, and woody, were defined as

405

the representative aroma attributes of Chios mastic gum by a panel consisting of 20 experienced

406

assessors. The intensities of the given odor attributes were rated and compared between Chios

407

mastic gum and the corresponding aroma model. The result of the aroma recombination study

408

demonstrated that the characteristic aroma of Chios mastic gum may well be simulated by

409

combining ten key odorants in their respective concentrations using water as matrix (Figure 3).

410

The performed one-way analysis of variance (p > 0.05) postulated no significant difference

411

between Chios mastic gum and the reconstituted aroma model. These data also revealed that the

412

key odorants of Chios mastic gum have been quantified accurately by MHS-SPME. Nevertheless, 20 ACS Paragon Plus Environment

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413

the intensity of the woody odor attribute was rated slightly lower in the reconstituted aroma

414

model, which might be caused by variable aroma release from different matrices (water and the

415

matrix of mastic gum) or by yet unquantified odorants with a woody note like -terpinene or

416

2-methoxytoluene. The applied headspace analysis may bring poor extractability for very polar

417

compounds as well as semi-volatiles,46 which may be an explanation for the lower rating

418

attributed to discrimination of some potential key odorants.

419

To gain deeper insights into potential interactions of potent odorants (OAVs > 1500) of Chios

420

mastic gum, omission experiments were carried out. Application of triangle tests revealed that the

421

odor impressions of two aroma models lacking -pinene or -myrcene, respectively, totally

422

differed from those of Chios mastic gum ( ≤ 0.1%) (Table 6). The omission of limonene,

423

-linalool and perillene from the complete aroma model also caused statistically significant

424

differences. The results obtained from omission tests confirmed the indispensable contribution of

425

-pinene, -myrcene, limonene, -linalool, and perillene to the overall aroma of Chios mastic

426

gum.

427

In conclusion, the characteristic aroma profile of Chios mastic gum was comprehensively

428

decoded for the first time by means of molecular sensory science. A broad range of potent

429

odorants were identified after HS-SPME and HS-SBSE and further quantified accurately by

430

means of MHS-SPME. Besides a major volatile (-pinene) a series of minor odorants, such as -

431

myrcene, limonene, -linalool, and perillene contributed to the typical aroma of Chios mastic

432

gum.



434

Supporting Information

435

Table S1. Key aroma compounds, flavor dilution factors (FD ≥ 8), and m/z fragments for

436

quantitation.

437

Table S2. Preparation of aqueous standard solutions for external calibration by means of MHS-

438

SPME.

439

Table S3. Odor-active compounds of Chios mastic gum perceived at the ODP after HS-SPME-

440

GC-MS/MS-O utilizing CAR/PDMS and CAR/PDMS fibers.

441

Table S4. Calibration parameters obtained for aqueous solutions of key aroma compounds of

442

Chios mastic gum quantified by means of MHS-SPME.

443

Figure S1. Influence of incubation time, extraction time, and extraction temperature on the

444

extraction efficiency of selected odorants expressed in percent of maximum peak area.


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445


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446

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581

Funding 28 ACS Paragon Plus Environment

Page 28 of 40

Page 29 of 40


582

The research (project number: SPFW-2018-YB07) was supported by the Open Project Program

583

of Beijing Key Laboratory of Flavor Chemistry ，Beijing Technology and Business University

584

(BTBU), Beijing 100048, China. HZ is grateful for financial support by the excellence initiative

585

of the Hessian Ministry of Science and Art which encompasses a generous grant for the LOEWE

586

focus “Aromaplus”.

587

Notes

588

The authors declare no competing financial interest.



Figure captions Figure 1. Enantiomeric ratios of -pinene (A), Z-verbenol (B), -pinene (C), and limonene (D) in Chios mastic gum. Figure 2. Linear plots (ln Ai against i-1) of selected odorants of Chios mastic gum. Figure 3. Comparative sensory analysis of Chios mastic gum and the reconstituted aroma model in water by the panel (n = 20).


Page 30 of 40

Page 31 of 40


Table 1. Odor-active compounds identified in Chios mastic gum by means of HS-SPME-GCMS/MS-O and HS-SBSE-GC-MS-O with TDU. RI No.

FD factor compound

odor impression

identification

< 900

acetone

fresh, solvent

< 900

< 900

ethanol

1026

924



1115d

950

944d

1093

1088d

992

990d

6

1126

1125

970



7

1138

1138

941

8

1171

1171

9

1199

10

1252

11b 12

VF-

WAXms

WAXms

Standard

1

< 900

< 900

< 900

2

935

935

3

1026

4a

1091

5a

HS-SPME

HS-SBSE

RI, odor, MS

2

-

ethanoic

RI, odor, MS

1

-

-pinene

forest-like, resinous

RI, odor, MS

256

128

E,E-5-caranol

green, forest-like, vanilla

RI, odor, MS

4

-

green, forest-like

RI, odor, MS

1

-

-pinenelit 7, 10-12

resinous, fresh, terpene-like

RI, odor, MS

128

32

942

camphenelit 7, 10-12

pungent, spicy, buttery

RI, odor, MS

64

8

994



-myrcene

pine-like, green, fresh

RI, odor, MS

-

128

1199

1025

1025

limonene

citrus-like, fruity

RI, odor, MS

8

-

1252

1062



-terpinenelit 10, 12

woody, green

RI, odor, MS

-

2

1300

1300

1020

1020

oct-1-en-3-one

mushroom-like

RI, odor

1

1

1389

1389

1089

1090

2-nonanonelit 12

green, fresh, resinous

RI, odor, MS

128

-

13

1408

1409

1000

1000

2-methoxytoluene

woody, fresh

RI, odor, MS

4

-

14c

1427

1427

1096

1096

perillenelit 7, 12

green, pea-like, citrus-like

RI, odor, MS

512

128

15a, c

1433

1435d

ndc



-campholenal

woody, green coffee-like

RI, odor, MS

2

1

16

1446

1452

< 900

< 900

acetic acid

sourish, vinegar-like

RI, odor, MS

2

1

17a

1492

1491d

1132

d

-campholenallit 7, 10, 11

green, fresh

RI, odor, MS

-

2

18

1546

1546

1095



-linalool

floral, fresh, fruity

RI, odor, MS

256

64

19a

1584

1584d

1151

1152d

E-verbenollit 7, 11, 12

earthy, hay

RI, odor, MS

-

8

20

1593

1593

nd

-

isothymol methylether

green, fruity

RI, odor, MS

4

-

21

1627

1627

1195

1195

myrtenal

pungent, sweaty

RI, odor, MS

4

-

22c

1642

1642

nd

-

terpinen-4-ollit 10, 12

earthy

RI, odor, MS

16

16

23

1675

1675

1147

1147

Z-verbenollit 7, 12

pine-like, sweetish

RI, odor, MS

8

-

24

1866

1866

1219

1219

carveollit 7, 10, 12

fresh, citrus-like

RI, odor, MS

2

-

sweetish, earthy, buttery

RI, odor, MS

8

-

a

d

DB-5ms

DB-5ms Standard

lit 7,10-12

6Z-2,6-dimethyl-2,6octadiene

lit 7, 10, 12

lit 7, 10-12

lit 7, 10, 12

lit 7, 11, 12

humulene-1,2-epoxide

lit

25a

2035

2035d

nd

-

26

2130

-

nd

-

unknown

sweetish, marzipan-like

-

4

-

27

2226

-

nd

-

unknown

earthy

-

1

-

7, 10,12

No authentic standards of the compounds were commercially available. The compounds were tentatively identified based on NIST MS database. MS spectrum was ambiguous. The compound was tentatively identified on the basis of RI, odor, and literature data15. c identified by means of HS-SBSE. d The retention indices were obtained from NIST Chemistry Webbook and Pherobase: Database of Pheromones and Semiochemicals. lit the compound was reported in the previous literature on Chios mastic gum.

a

b



Page 32 of 40

Table 2. Enantiomeric ratios and ee values of key chiral odorants of Chios mastic gum. compound -pinene -pinene camphene limonene -linalool terpinen-4-ol Z-verbenol

ratio (R/S, %) 99.6 / 0.4 63.7 / 36.3 73.0 / 27.0 37.8 / 62.2 4.3 / 95.7 78.4 / 21.6 0.8 / 99.2


ee (%) 99.2 ± 0.04 27.4 ± 5.16 46.0 ± 4.53 25.3 ± 6.31 91.3 ± 0.51 56.1 ± 0.58 98.3 ± 0.98

Page 33 of 40


Table 3. Correlation coefficients (R2) for ln Ai against i-1 of key aroma compounds using different masses of mastic gum. compound

-pinenea

-pinene camphene -myrcene limonene 2-nonanone perillene -linalool terpinen-4-ol Z-verbenolb

10 NL 0.935 0.976 0.982 0.995 0.948 0.991 0.984 0.977 NL

mass in HS vial (mg) 20 30 NL NL 0.985 0.982 NL NL 0.929 0.995 0.973 0.951 0.978 0.970 0.993 0.956 0.995 0.910 0.954 NL NL NL

40 NL NL NL 0.971 0.927 0.998 0.958 0.998 NL 0.921

NL not linear. a The compound showed good linearity (R2 = 0.962) when amount of sample was decreased to 1 mg. b The compound showed a good linearity (R2 = 0.952) when the amount of sample was adjusted to 35 mg.



Page 34 of 40

Table 4. Parameters (A1*, ln β, β & AT) measured for quantitation of key aroma compounds of Chios mastic gum. compound -pinene -pinene camphene -myrcene limonene 2-nonanone perillene -linalool terpinen-4-ol Z-verbenol

mass (mg) 1 10 10 10 10 10 10 10 10 35

A1*

area

intercept

peak

of

first

A1*

ln β

β

AT

15000000 ± 4000000

-0.11 ± 0.042

0.90 ± 0.042

150000000 ± 40000000

2000000 ± 330000

-0.46 ± 0.068

0.76 ± 0.043

5700000 ± 600000

64000 ± 22000

-0.28 ± 0.096

0.76 ± 0.073

270000 ± 58000

9300000 ± 1900000

-0.47 ± 0.083

0.63 ± 0.051

25000000 ± 3600000

2200000 ± 490000

-0.38 ± 0.087

0.69 ± 0.058

7100000 ± 910000

140000 ± 37000

-0.59 ± 0.055

0.56 ± 0.030

320000 ± 75000

1400000 ± 460000

-0.59 ± 0.085

0.56 ± 0.045

3100000 ± 740000

2000000 ± 140000

-0.44 ± 0.061

0.66 ± 0.039

5500000 ± 640000

64000 ± 13000

-0.25 ± 0.042

0.78 ± 0.032

290000 ± 19000

810000 ± 59000 extraction.

β

constant

-0.10 ± 0.010 for

the

extractability


0.90 ± 0.009 of

each

compound.

8500000 ± 120000 AT

total

peak

area.

Page 35 of 40


Table 5. Concentrations, odor thresholds, and odor activity values (OAVs) for key aroma compounds detected in Chios mastic gum. compound -pinene -pinene camphene -myrcene limonene 2-nonanone perillene -linalool terpinen-4-ol Z-verbenol a

concentration (µg/g) 10000 ± 2500 80 ± 4.0 17 ± 1.3 200 ± 9.0 99 ± 8.0 49 ± 0.3 130 ± 31 44 ± 5.1 1.9 ± 0.1 230 ± 29

odor threshold (µg/g) 0.00623 0.1423 0.45a 0.01324 0.1025 0.2025 0.06526 0.00627 0.04128 0.30a

determined in this study.


OAV 1700000 ± 420000 570 ± 29 38 ± 3 15000 ± 680 9900 ± 792 250 ± 2 2000 ± 460 7300 ± 880 46 ± 2 770 ± 100


Page 36 of 40

Table 6. Omission experiments from the complete aroma model odorant omitted from the complete model mixture -pinene -myrcene limonene -linalool perillene

na

significance (α, %)b

18 15 11 11 12

≤ 0.1 ≤ 0.1 ≤ 5.0 ≤ 5.0 ≤ 5.0

a number of correct judgments from 20 assessors evaluating the aroma difference by means of a triangle test. b significance value (%): ≤ 0.1 very highly significant, ≤ 1.0 highly significant, ≤ 5.0 significant, and ≥ 5.0 not significant.


Page 37 of 40


Figure 1.



Figure 2.


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Page 39 of 40


Figure 3.



Table of Contents Graphics (TOC graphic)


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Aroma Investigation of Chios Mastic Gum (Pistacia lentiscus var. Chia

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